There is a continuing need in medical practice and research, and in analytical and diagnostic procedures for rapid and accurate determinations of chemical and biological substances which are present in various fluids, such as biological fluids. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, metabolities, toxins, viruses, microorganisms or nucleic acids in human or animal body fluids or tissues must be determined rapidly and accurately for effective research, diagnosis or treatment.
In approximately the last twenty years, a wide variety of analytical methods have been developed to detect the substances noted above. Generally, the state of the art has advanced to such a degree that analytical and diagnostic methods have become highly reliable and suitable for automation or for use with test kits which can be readily used in doctor's offices or at home. Most of such methods rely on what are known in the art as "specific binding" reactions in which an unknown substance to be detected (known as a "ligand") reacts specifically and preferentially with a corresponding "receptor" molecule. Most well known specific binding reactions occur between immunoreactants, such as antibodies and antigens (foreign substances which produce immunological responses), but other specific binding reactions (such as between avidin and biotin, and a sugar with a lectin) are well known.
Methods in the art using specific binding reactions generally require that one or more or both of the reactants be immobilized on a solid substrate of some type, so that unreacted (and generally water-soluble) materials can then be separated from the water-insoluble reaction product (often called a "complex"). In addition, such immobilized reactants can be used in affinity chromatography to remove a desired biologically active material from a mixture of such materials.
Biologically active substances have thus been immobilized to advantage on particulate substrates such as polymeric particles, animal and human erythrocytes, bacterial cells and other materials known in the art. For example, carrier particles prepared from epoxy-group containing monomers are described in U.S. Pat. No. 4,415,700 (issued Nov. 15, 1983 to Batz et al). Carboxylated latex particles have also been used to prepare diagnostic reagents, as noted in U.S. Pat. No. 4,181,636 (issued Jan. 1, 1980 to Fischer). Where polymeric particles have been used as carrier substrates, biologically active substances have been attached through reactive groups on the particle surface, such groups provided either from the polymer composition or from linking moieties attached to the particles. U.S. Pat. No. 4,401,765 (issued Aug. 30, 1983 to Craig et al) describes a number of reactive groups on polymeric particles.
Several advances in the art in this regard are described in U.S. Ser. No. 081,206 (filed Aug. 3, 1987 by Sutton et al), U.S. Ser. No. 136,165 (filed Dec. 18, 1987 by Burdick et al) and EP-A-0 308 235 (published Apr. 26, 1989 and corresponding to U.S. Ser. No. 373,304, filed Jun. 29, 1989 by Sutton et al as a CIP of U.S. Ser. No. 098,429, filed Sep. 18, 1987, now abandoned). These applications describe various means for attaching biologically active substances to polymeric particles having various reactive surface groups.
U.S. Pat. No. 3,983,001 (issued Sep. 28, 1976 to Coupek et al) describes the use of hydrophilic macroporous copolymers as carriers for biologically active compounds in the preparation of affinity chromatography reagents. Such copolymers can be prepared from certain hydrophilic monomers such as polyglycol acrylates and methacrylates. Biologically active compounds are adsorbed to the carrier polymers. Absorption, however, does not provide for optimum sensitivity or efficiency in affinity chromatography.
The modification of protein adsorption on polymeric surfaces has been a common goal for many workers trying to apply polymer technology to in vivo and in vitro uses in biotechnology. Undesirable protein adsorption has been a continual problem. For example, nonspecific adsorption is a major concern in the use of polymers for affinity chromatography for purification of proteins.
The modification of polymer surfaces has taken many forms, including physical coatings, graft copolymerization, chemical treatments and plasma gas discharge treatment. The hydrophilic nature of the polymer surface has been the subject of considerable debate and research because an increase in hydrophilicity reduces adsorption of some proteins, but not others. As noted in the art cited above, the use of reactive side chains has also received considerable attention in the art. However, if the polymer particles are too hydrophilic and swell in aqueous solutions (as in U.S. Pat. No. 3,983,001, noted above), the assays can be adversely affected.
One technique commonly used to reduce nonspecific adsorption of proteins is what is called "capping". After a desired protein (for example, an antibody) is covalently attached to polymeric particles, another nonimmunoreactive protein is allowed to adsorb to the particle surface to "cap" the remaining reactive sites. While this is generally effective in some cases, it would be desirable to avoid this step because of the expense and extra time it requires for preparing useful reagents. Thus, there is a continuing need for a means to provide biological reagents comprising polymeric particles as carriers without the need for "capping" and where nonspecific interactions are considerably reduced or eliminated entirely.